Treponema denticola is a Gram-negative human spirochete indigenous to the oral cavity; it has been recognized as one of three key bacterial species from the red complex bacterial community that is strongly associated with onset and progression of periodontitis. Periodontal disease is prevalent in humans, and has been associated with a variety of systemic diseases including cardiovascular disease, and preterm birth. However, little is known of T. denticola virulence, as the organism is difficult to grow and the genetic transformation system is not well established. Our goal is to identify virulence factor involved in T. denticola pathogenesis. One of the major virulence attributes that has been extensively studied in Gram-negative bacteria is lipopolysaccharide (LPS). There has been controversy over the issue of whether oral treponemes contain LPS. Despite previous investigations regarding LPS activity in T. denticola outer membrane (OM), a recent structural study has determined that T. denticola possesses a new type of OM lipid, different from LPS. Genomic analysis of T. denticola has also revealed that none of the genes essential for synthesis of classical LPS exist in the genome. The new type of OM lipid has been classified as a lipooligosaccharide (LOS). LOS is a prominent surface component of T. denticola, whose structure is very similar to the structural motifs found in lipoteichoic acid, an important Gram-positive bacteria virulence factor. In vitro studies using purified LOS have revealed T. denticola LOS can enhance osteoclastogenesis and alter soft tissue integrity. To date there has been no effort to evaluate the importance of differentially modified LOS in bacterial virulence in vivo as no defined mutants have been constructed to delineate the LOS biosynthetic pathway. Importantly, we have made progress in the genetic transformation of T. denticola, and have identified a genetic locus that is potentially responsible for the synthesis and assembly of T. denticola LOS. Furthermore, we have generated a TDE1418 mutant for a gene that encodes a putative K5 antigen synthase in the locus; the TDE1418 mutant impaired modification of LOS. Interestingly, the TDE1418 mutant failed to promote bone resorption in a mouse model for endodontic infection, suggesting T. denticola LOS is involved in bacterial virulence in vivo. We are now in a position to determine the genetic basis of LOS biosynthesis and examine its biological activity; thereby testing our hypothesis that the LOS structure assembled by T. denticola mediates bacterial virulence. We will achieve this goal by carrying out two specific aims: (1) Determine how LOS biosynthesis and assembly is modulated in the TDE1418 mutant. (2) Assess the functional contribution of LOS to T. denticola-induced bone resorption in vivo-utilizing the TDE1418 mutant. This proposal will permit us to further develop genetic transformation protocols, and investigate uncharacterized virulence factors such as LOS that will help define the role of oral spirotchetes in the pathogenesis of periodontal and endodontic diseases.